Diseases of the bone marrow that may be treated with cryopreserved hemopoletic stem cell transplantation indude the leukemias, lymphomas and aplastic anemia. In the field of cryopreservation, current opinion is that cryoprotective agents should be removed prior to transplantation. In the case of dimethylsulfoxide (DM80), the most popular cryoprotectant, there are many documented effects at the cellular level along with frequent clinical reports of detrimental side effects in patients. The ability to use non-cytotoxic sugars should enable the avoidance of DMSO in currently employed cryopreservation procedures. Therefore, in this proposal we will use human stem/progenitor cells to develop preservation methods utilizing a new cell membrane permeabilization technology in combination with sugars that are normally impermeable. It is anticipated that cells cryopreserved by this technology will be directly infusible into patients. This technology may also be leveraged to provide stable long-term storage of a variety of cells, including mature blood cells, mesenchymal stem cells, cell-based biosensors and some medical therapies involving gene therapy. The following specific aims will be addressed in this Phase I research study: #1 Optimization of H5 pore and divalent metal ion (zinc for pore opening and dosing) concentrations; #2 Optimization of postpermeabilization pore removal; #3 Comparison of controlled rate cryopreservation at four cooling rates following permeabilization with trehalose or sucrose; and #4 Evaluation of residual H5 cytotoxicity and the long-term hematopoietic potential of treated cells. These aims will be accomplished using human CD34+ cord blood cells and a panel of in vitro assays, and by transplantation in vivo into NOD/SCID mice. The technical innovations in this proposal are based upon the concept of reversible permeabilization of cell membranes using engineered pores to load the cells with disaccharides prior to freezing. The permeabilization technology employs a genetically engineered pore forming protein. The opening and dosing of the pore is regulated by divalent metal ions. Preliminary feasibility data is submitted demonstrating cryopreservation of a porated human hemopoietic progenitor cell line with trehalose. A Phase-Il SBIR study wilt be proposed to optimize hemopoletic stem and progenitor cell preservation if mice treated with porated human cells demonstrate good survival and hematopoietic reconstitution.